JP4688667B2 - Scanning imager and imaging method for specimen imaging - Google Patents

Description

  The present invention relates to an imaging technique. The present invention can be used in various imaging systems, but its main application is low density / high density cell detection, localization and identification in blood smears, biological samples and the like. Therefore, although the present application will describe an example in which the present invention is applied to this application, it should be understood that the present invention has other applications. The present invention relates to various technologies existing on a substantially flat surface or a sample in the fields of science and technology such as printing engineering, electronic engineering, medicine, etc. Can be used to image, locate and identify low density / high density objects.

  A problem inherent to special cell (RC) research is that the RC concentration in blood and other body fluids is usually low. In RC research, usually, a body fluid such as blood is first treated to remove unnecessary cells therein, and a fluorescent substance that adheres to the antibody is added to selectively attach the antibody to the RC surface or RC protein. The attached RC protein is a membrane protein or an intracellular protein such as a cytoplasmic protein, and this antibody also adheres to other molecules and DNA in the RC.

The fluorescent substance to be added may be any substance that can identify the cell of interest, such as a fluorescent marker dye. For example, when identifying a target type of RC cell from cells contained in blood or a smear containing its constituents, the smear is optically analyzed after processing in the manner described above. That's fine. At that time, it is important to obtain a statistically accurate analysis result to obtain more cells than necessary for the analysis process, but this is difficult. For example, in an RC study involving one RC per million cells, if you need to identify at least 10 RCs for statistical accuracy, sample at least 10 million cells However, the area of a blood smear containing more than 10 million cells is usually 100 cm ² . As you can see, the required number of cells shown here is only an example, and depending on the type of test or study, statistical accuracy may not be achieved without obtaining more cells than this example. The numerical value of one RC per million cells is only an example to show the rareness of RC that can be observed, and the concentration may be lower than this. The invention described in the present application can be applied to cases where the required number of cells is larger or smaller than this example, and also when the cell concentration is higher or lower than this concentration example. Not only the identification but also a known or later-developed cell identification method using a quantum dot or a nanoparticle probe as the identification means.

US Pat. No. 4,600,951 U.S. Pat. No. 4,849,645 U.S. Pat. No. 4,875,780 U.S. Pat. No. 4,941,309 US Pat. No. 5,017,798 US Pat. No. 5,216,485 US Pat. No. 5,471,662 US Pat. No. 5,627,365 US Pat. No. 5,640,246 US Pat. No. 5,798,831 US Patent Application Publication No. 2002/186368 (A1) US Pat. No. 6,545,334 (B2) US Pat. No. 5,892,577 US Pat. No. 4,721,851 U.S. Pat. No. 4,010,364 U.S. Pat. No. 4,0028,29 US Pat. No. 5,732,162 US Patent Application Publication No. 2004/71330 (A1) US Patent Application Publication No. 2004/71332 (A1) US Patent Application Publication No. 2004/131241 (A1) British Patent No. 1579188 JP-A-4-296642 Japanese Patent Laid-Open No. 6-148085 JP-A-9-145631 Diana W. Bianchi et al., "Fetomaternal Cellular and Plasma DNA Trafficking", The Yin and The Yang, Annals New York Academy of Sciences, pp.119-131 Josh Wolfe, "A Thousand Dots of Light", Forbes / Wolfe Nanotech Report, May 29, 2002, [online] Internet URL: http://www.forbes.com/ Barbara M. D. Pertl et al., "Fetal DNA in Maternal Plasma: Emerging Clinical Applications", The American College of Obstetricians and Gynecologists, Published by Elsevier Science Inc., Vol. 98, No. 3, September 2001, pp.483-490 Kenneth D. Bauer et al., "Reliable and Sensitive Analysis of Occult Bone Marrow Metastases Using Automated Cellular Imaging", Clinical Cancer Research, Vol.6, pp.3352-3359, September 2000 Thomas E. Witzig et al., "Detection of Circulating Cytokeratin-positive Cells in the Blood of Breast Cancer Patients Using Immunomagnetic Enrichment and Digital Microscopy", Clinical Cancer Research, Vol.8, pp.1085-1091, May 2002 R. A. Ghossein et al., "Molecular Detection and Characterization of Circulating Tumour Cells and Micrometastases in Solid Tumours", European Journal of Cancer, 36 (2000), pp.1681-1694, March 2000, Elsevier Science Ltd. Kjersti Flatmark et al., "Immunomagnetic Detection of Micrometastatic Cells in Bone Marrow of Colorectal Cancer Patients", Clinical Cancer Research, Vol.8, pp.449-449, February 2002 G <bor Mihes et al., "Quantitative Analysis of Disseminated Tumor Cells in the Bone Marrow by Automated Fluorescence Image Analysis", Cytometry (Communications in Clinical Cytometry), 42: 357-362, 2000, Wiley-Liss, Inc. M. Werther et al., "The Use of the CELLection Kit in the Isolation of Carcinoma Cells from Mononuculear Cell Suspensions", Journal of Immunological Methods, 238 (2000), pp.133-141, 2000 Elsevier Science B. V. S. A. Burchill et al., "Comparison of the RNA-Amplification Based Methods RT-PCR and NASBA for the Detection of Circulating Tumour Cells", 2002 Cancer Research Campaign, British Journal of Cancer (2002) 86, pp.102-109

  Therefore, it is possible to realize high-speed scanning of a large amount of cells, which is a key for improving the throughput of the test process, that is, a cell detection system or a system capable of increasing the cell detection speed in the process (more preferably, the reliability of the cell detection process ( It can be said that there is a need to provide a system that can improve accuracy and reduce costs.

  In order to meet such a demand, many cell detection techniques have been proposed so far. For example, FISH (fluorescence in situ hybridization), flow cytometry, LSC (laser scanning cytometry) and the like.

  These systems are all systems that have been developed with the aim of improving the scanning speed, but the FOV (field of view) is somewhat narrower than that of a microscope, and the target scanning speed is realized. It has not reached.

  In view of these problems, US Patent Application No. 10/271347, the entire contents of which are incorporated herein by reference, proposes a technique called FAST (fiber array scanning technology). FAST is a technique that can increase the speed at which a specimen is scanned and thus the speed at which cells that appear to be RC are detected, thus allowing examination of large specimens. Also, the speed improvement effect by the technique described in the above-mentioned US application can be enhanced by taking appropriate measures. For example, a plurality of sets of light sources and signal detectors may be provided. More specifically, a plurality of light sources (more generally radiation sources; the same applies below), for example, lasers that emit excitation light (more generally excitation fields or radiation beams; the same applies hereinafter) having different wavelengths or wavelength ranges. In addition to providing a light source, a plurality of signal detectors adjusted or calibrated so as to detect fluorescence emitted from the specimen by irradiation of excitation light from the corresponding light source are provided. It is a means of operating a signal detector simultaneously. When implementing this configuration, each signal detector could be provided with a filter so that only the corresponding fluorescence to be detected is detected and captured as an optical signal. By adopting such a configuration, the amount of information that can be detected in one scan is basically N times.

  However, this configuration still has problems. First, the fluorescence signal stimulated and emitted by the excitation light from a certain light source and the fluorescence signal stimulated and emitted by the excitation light from another light source may considerably overlap in a considerable part in wavelength. This problem can be alleviated if filtering is performed in a wavelength selective manner. However, the wavelength range suppressed by the filtering is a useful wavelength range that should have been included in the detection target, and it is disadvantageous that such a wavelength range is suppressed. Second, when implementing the above-described configuration, a beam splitter or dichroic mirror is placed on the optical path of the emitted fluorescence signal, and the propagation destination of the desired component in the fluorescence signal is used as the desired signal detector. Although it may be appropriate to adopt a configuration in which the light is directed, if such a configuration is adopted, the detected fluorescence signal intensity is further reduced.

A scanning imager for specimen imaging according to an embodiment of the present invention includes an imager stage that supports a specimen, an entrance-side opening that is disposed close to the specimen so as to capture the specimen on the imager stage within its field of view, and a distance from the entrance side opening. an optical path having an exit-side opening at a location with a plurality of radiation sources that emit placed by and respectively the radiation beam is divided into a plurality based on the input elevation side opening, has two or more reflective surfaces, each reflective surface includes a rotating polygon scanner査run on the selected area of the sample was in the radiation reflected radiation beam incident from the radiation source corresponding to the respective reflecting surfaces,
Light detection that occurs due to the action of the radiation beam on the specimen, is received at the entrance-side aperture, transmitted to the exit-side aperture via the optical path, and received and detected by associating the light signal emitted from the exit-side aperture with each radiation source A scanning imager for specimen imaging , comprising: a detector system; and a processor that processes an optical signal detected by the photodetector system in association with each radiation source , wherein each radiation source corresponds to each radiation source The radiation beam reflected by each reflecting surface of the rotating polygon scanner emits, and the reflected radiation beam is shifted in the direction perpendicular to the scanning direction of each radiation beam. The scanning angle can be shifted in order, and the angle of incidence on the rotating polygon mirror is such that the number of scans of the sample per rotation of the rotating polygon scanner is increased. That are arranged in a rotating polygon mirror and a predetermined positional relationship, characterized by.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. It should be noted that the present invention can be implemented by variously combining various components or steps, and the constituent elements of the embodiment described below and how to combine them are merely examples shown for explanation.

  FIG. 1 shows an example of an imaging apparatus or an imager for the purpose of understanding the embodiment of the present invention. In the present application, this imager will be described first, and then various embodiments of the present invention, improvement of the scanning speed by them, and a technique for solving the above-described problems in them will be described. The imager 10 shown in FIG. 1 is a type of imager that performs laser beam scanning with a galvanometer. The number of laser light sources provided is one (62), and the number of signal detectors is one (90). is there. As shown in the figure, this imager 10 is an imager used for examining a specimen 12 with a biological smear 14 or the like on a (partial) surface of a slide 16, for example. That is, it is designed so that a minute or microscopic object can be detected as shown in an enlarged manner later.

  As known in this technical field, when preparing a specimen 12 for cell research, for example, a cell monolayer, a body fluid specimen such as blood or its components is extracted from the subject, and the body fluid specimen such as a marker dye is extracted from the body fluid specimen. It is desirable to treat with a fluorescent material. A substance used as a fluorescent substance is a substance that selectively adheres to various biomolecules such as proteins and nucleic acids existing on the surface or inside of cells. In this technical field, several types of fluorescent substances that can be suitably marked on various cells of clinical interest, such as certain cancer cells that need to be examined, blasts, etc., are known, Efforts are also being made to develop marking materials for a wide variety of cells such as brain cells, hepatocytes, and bacterial cells. A fluorescent material used for marking emits characteristic light such as fluorescence or phosphorescence when receiving an intended excitation radiation (radiation of light having a predetermined wavelength to a predetermined spectral distribution, X-ray, electron beam, or the like). This characteristic emission usually has a unique wavelength or a spectral distribution over a unique wavelength range. In addition, although marking with a dye is a typical tagging process, there is a technique using a marker known as a quantum dot, a DNA nanoparticle probe, or the like.

  The specimen 12 created by applying the smear 14 on the slide 16 is mounted on a transfer imager stage (slide holder) 20. Although only a part of the stage 20 is shown in FIG. 1, the stage 20 includes a track 22 and a motor 24. The track 22 is a track that can be linearly moved, and the specimen 12 is supported by the track 22. The motor 24 is connected to the track 22 via a gear 26. When the motor 24 is operated, the track 22 and the specimen 12 supported by the track 22 are in the y direction indicated by an arrow 28 in the figure. And it will be transferred along the x direction shown by the arrow 29 in the figure. In FIG. 1, the linear transfer type track 22 is driven by the rotary motor 24, but other types of driving mechanisms may be used. Further, instead of or in addition to the linear movement, other types of movement such as rotating the specimen 12 may be performed.

  As shown in FIGS. 1 to 3, the imager 10 has a first end 42 and a second end 44 as an optical path for transmitting an optical signal from the incident side opening 48 to the emission side opening 52. A fiber bundle 40 is provided. The fiber bundle first end 42 is at a location near the sample 12, and the fiber bundle second end 44 is at a location away from the sample 12. As best seen in FIG. 3, the fiber bundle first end portion 42 has a plurality of individual fiber first ends 46 that are substantially parallel to each other. In addition, an incident-side opening 48 that is long in the x direction is formed side by side in a shape that can be referred to as a substantially linear or high aspect ratio square. It is desirable to increase the number of the individual fiber first ends 46 forming the incident side opening 48 parallel to the sample 12, that is, the number of fibers, for example, several thousand or more. For example, 40,000 fibers having a diameter of about 50 μm may be used and arranged in an array of 40 × 1000. The incident-side opening 48 formed thereby has a shape / dimension of about 5 cm in the major axis direction and about 0.2 cm in the minor axis direction, and thus its aspect ratio is 25: 1. The arrangement of the individual fiber first end portions 46 may be a regular pattern as shown in FIG. 3, but may be arranged irregularly or in an array with an aperiodic pattern. The thickness of the fiber may be thicker or thinner than 50 μm. The illustrated fiber is a fiber having a substantially circular end 46, but a fiber having another cross-sectional shape such as an egg shape, a quadrangle, a hexagon, or the like may be used. In any case, the individual fiber first end portion 46 is opposed to the smear 14 in a direction as close to the orthogonal direction as possible with respect to the substantially flat surface on which the smear 14 is applied so that the smear 14 enters the field of view. .

  As best seen from FIG. 2, the optical fiber bundle 40 is shaped. That is, the cross-sectional diameter and cross-sectional shape of the optical fiber bundle 40 change from the first end 42 to the second end 44. The second end portion 50 of a plurality of fibers appears in the fiber bundle second end portion 44, but these individual fiber second end portions 50 are gathered together with shaping to form a substantially circular emission side opening 52. Forming. Since the individual fiber first end 46 and the individual fiber second end 50 are both ends of the same individual fiber, there is a one-to-one correspondence between them, and there is a corresponding first end 46 and this. The fiber connecting the second end 50 is a separate fiber from the fiber connecting the other first end 46 and the corresponding second end 50. Each fiber is preferably a fiber having its own waveguide cladding, but it has only a light-transmitting fiber core and does not have a cladding, but has an optical waveguide at the interface between the atmosphere and the core. A fiber that performs a function may be used. In addition, other types of optical fibers, for example, fibers well known in the present technical field can also be used. Typically, various fibers may be mentioned that are manufactured by extrusion and formed from glass, plastic or other light transmissive material. In FIG. 2, the dashed lines show the paths of two separate fibers 56 and 58. The optical fiber bundle 40 shown in this figure is shaped so as to converge from the spread substantially straight first end portion 42 to the substantially circular second end portion 44. For example, as described above, in the example in which 40,000 fibers having a diameter of 50 μm are used, the emission side opening 52 is circular with a diameter of about 1.3 cm. Such shaping can be suitably realized by continuously changing the spatial positional relationship between the fibers in the optical fiber bundle 40 (for example, 56 and 58).

  It should be noted that the spatial relationship between the individual fiber first end 46 and the individual fiber second end 50 can be determined approximately arbitrarily. The fibers 56 and 58 illustrated in FIG. 2 originate from approximately the same location in the entrance aperture 48, but the fiber 56 terminates at approximately the top of the exit aperture 52, whereas the fiber 58 is approximately in the middle. It ends with. In consideration of the fiber arrangement, a predetermined correspondence between the position of the individual fiber first end 46 in the incident side opening 48 and the position of the individual fiber second end 50 in the output side opening 52 is provided. However, there is no proper correspondence between the positions of the end portions 46 and 50, and an arbitrary positional relationship may be used as a whole. A shaped optical fiber bundle similar to the shaped optical fiber bundle 40 is known and used for other purposes in the field of optical technology. For example, it is used for converting focused light into a linear irradiation pattern, or used for introducing a light beam into a linear slit of a spectroscope or spectrometer.

  In order to improve the optical signal transmission efficiency from the entrance side opening 48 to the exit side opening 52, the fiber binding density in the optical fiber bundle 40 should be increased, for example, a coefficient of about 0.80 or more. desirable. As factors affecting the optical signal transmission efficiency, in addition to the optical fiber bundling density, the tip end polished state of the individual fiber first end 46 and the second end 50 or the light transmission characteristics, the fiber (56, 58, etc.) Absorption rate per unit length, total length of fibers (56, 58, etc.), and the like. Further, the fiber bending loss can be suppressed well by preventing the bending of the optical fiber bundle 40 from becoming steep as shown in FIGS. 1 and 2, that is, by bending the optical fiber bundle 40 gradually. Can do. By gradually bending in this manner, it is possible to change the opening directions of the entrance side opening 48 and the exit side opening 52 as shown in FIG.

  The capture optical transmission form of the optical fiber bundle 40 is shown here. However, as long as good characteristics can be obtained, various optical transmission elements, optical paths or light pipes that are known or will be developed in the future are used. You will understand that it can be used as a pipe.

  As shown in FIGS. 1 to 3, in this reference example, a scanning light source composed of a laser light source 62 is used as the scanning radiation source 60. The laser light source 62 generates excitation light 64 that is a radiation beam in a predetermined wavelength or wavelength region. The wavelength or wavelength range is set to a wavelength or wavelength range that can excite a substance used for marking on the smear 14. In addition, a galvanometer 66 is provided as means for angularly scanning the specimen 12 from the crossing direction by the radiation beam 64. The galvanometer 66 has a reflecting surface, and this reflecting surface rotates as indicated by a curve 68 with an arrow in the drawing in accordance with an electric input to the galvanometer 66. Further, a focusing lens set 70 may be provided to focus the radiation beam 64 that has been scanned at an angle on the specimen 12, particularly the smear 14. Whether the focusing lens set 70 is present or not, when the reflecting surface of the galvanometer 66 is repeatedly rotated, the direction of the radiation beam 64 is repeatedly changed, as indicated by a straight line 72 with an arrow in the figure. The smear 14 is linearly scanned or scanned at high speed by the radiation beam 64 from the crossing direction along the linear locus 74 below the incident side opening 48. The scanning light source 60 and the optical fiber bundle 40 are configured or arranged so that the linear locus 74 is directly below the incident side opening 48 and parallel to the major axis direction of the incident side opening 48. That is, in the coordinate system shown in FIG. 1, the direction of the linear locus 74 is set to the x direction. It may be said that the smear 14 is positioned approximately 1 mm below the incident side opening 48 and the linear locus 74 is generated in a portion of the smear 14 immediately below the incident side opening 48. Of course, depending on what apparatus and in what environment this conceptual configuration is implemented, the preferred distance (positional deviation) of the incident-side opening 48 with respect to the linear locus (scanning axis) 74 is different. become.

  When used for cell research, it is desirable that the size of the irradiation spot formed on the smear 14 by the radiation beam 64 for excitation is approximately the size of the cell. Although the size of the irradiation spot or cell varies depending on the case, it is usually about 1 to 30 μm. In order to focus on such a narrow region, it is better to focus the beam using the focusing lens set 70.

  The electronic control unit 80 as a processor shown in FIGS. 1 to 3 supplies signals to and receives signals from the galvanometer 66 and the transfer imager stage 20 (motors thereof). Through operation, the sweep or scan operation (72) along the linear locus 74 by the radiation beam 64 and the transport operation of the specimen 12 are coordinated, thereby performing a transverse raster scan for a predetermined area on the specimen 12. At this time, the x-direction scanning width is limited and defined by the shorter one of the length of the linear locus 74 and the long axis direction dimension of the incident side opening 42, so the length of the linear locus 74 is limited to the long axis direction of the incident side opening 42. It is good to match the dimensions.

  The excitation radiation beam 64 is incident on the smear 14 from an oblique direction at an angle larger than the condensing angle θ to the incident side opening 42. The condensing angle θ may vary depending on the short-axis direction dimension of the incident side opening 42, the distance between the incident side opening 42 and the smear 14, and the condensing characteristic of the individual fiber first end 46. A characteristic value that suitably represents the condensing characteristic listed last is the numerical aperture of the first end 46 of the individual fiber. As is known in this technical field, it is usually beneficial to increase the light collection angle θ in order to collect the weak characteristic light emission from the cells, so use an optical fiber with a large end numerical aperture. Is normal. When the condensing angle θ is set to be large, the incident angle of the radiation beam 64 on the sample 12 needs to be increased, so that it is set to 30 to 90 °, for example. Means for allowing the radiation beam 64 to be incident on the specimen 12 at about 90 ° include, for example, U.S. Application No. 11/017440, which is hereby incorporated by reference in its entirety, and correspondingly with this application. There is a configuration disclosed in a Japanese application filed at the same time, in particular, an optical path with a branching portion that can collect light on both sides of a locus formed by scanning of a radiation beam.

  One reason for making the incident angle of the radiation beam 64 larger than the condensing angle θ by the incident side opening 42 is to prevent the radiation beam 64 light reflected by the mirror surface from being collected by the incident side opening 42. is there. Nevertheless, a significant part of the characteristic luminescence is collected by the entrance-side aperture 42 because the cells that have been subjected to the marking process normally operate as a point light source that emits characteristic luminescence spatially isotropically. This is because the incident-side opening 42 is arranged along and in close proximity to the locus 74 formed on the smear 14 by the radiation beam 64 and the numerical aperture of the individual fiber first end 46 is large. Characteristic light emission from the cells thus enters the individual fiber first end 46 in the incident side opening 42 and is collected, and then the individual fiber corresponding to the individual fiber first end 46 (for example, 56 in FIG. 2). , 58) and transmitted as an optical signal to the individual fiber second end 50 associated with the fiber, where it is emitted from the optical fiber bundle 40.

  Also, as can be appreciated, characteristic luminescence emitted from a particular cell is not collected by all or most of the multiple individual fiber first ends 46. Usually, one or several individual fiber first ends 46 in the immediate vicinity of the cell collect light. For example, the radiation beam 64 is irradiated so that the size of the irradiation spot to be formed is approximately the same size, for example, 10 to 15 μm, according to the size of the cell to be detected. On the other hand, since the diameter of the first end portion 46 of the individual fiber is, for example, about 50 μm, it is necessary to capture and collect the characteristic luminescence generated in any cell in the field of view in accordance with the swept radiation beam 64. Only one or a few fibers are required per cell.

  On the other hand, at the second end portion 44 of the optical fiber bundle 40, the individual fiber second end portion 50 is arranged so as to form a small output side opening 52. Therefore, the characteristic light collected by the individual fiber first end 46 corresponds to the individual fiber first end 46 regardless of which individual fiber first end 46 has collected the characteristic light emission. It is emitted from a small space area. As the radiation beam 64 is swept following a linear locus 74 formed immediately below the incident side opening 42 and parallel to the incident side opening 42, and a cell emitting characteristic light emission is changed to another cell accordingly. The characteristic light emission is collected from one or several individual fiber first end portions 46 located close to the previous characteristic light emission cell, and one piece located near the next characteristic light emission cell. Or it is taken over to several individual fiber first ends 46. The feature light thus collected is transmitted to the compact exit side opening 52 by the optical fiber bundle 40.

  The characteristic light transmitted to the emission side opening 52 as an optical signal is emitted from the emission side opening 52 and enters the signal detector 90. The signal detector 90 is provided in the vicinity of the emission side opening 52 and is a light-shielding filter disposed between the first lens 92 and the second lens 96 in addition to the first lens 92 that collimates the light from the emission side opening 52. 94. The light blocking filter 94 may be an interference filter whose reflectance peak wavelength is coincident with the center wavelength of the radiation beam 64 so that light from the scanning radiation source 60 is not incident on the photodetector set 98, for example. . As is known in the technical field, the unnecessary light removal ratio by the optical interference filter strongly depends on the incident angle of light. For example, the interference filter used when actually manufacturing this configuration has a removal ratio of 106: 1 or more when the deviation angle of the incident direction with respect to the orthogonal direction is within ± 14 °. In order to cope with this, a lens group is used as the first lens 92 that collimates the light emitted from the emission-side opening 52 while suppressing the divergence angle within ± 10 °. Such a lens group can be designed by using a conventionally known optical design method.

  As shown in FIG. 1, the signal detector 90 includes a second lens 96 that focuses the light collimated by the first lens 92 onto the photodetector set 98. Since these focusing optical systems (92 and 96) are attached to the compact exit side opening 52 and focused on the photodetector set 98 of the signal detector 90, the entrance side opening 42 is linear. A signal detection result can be obtained by the photodetector set 98 with respect to the characteristic luminescence incident on the incident side opening 42 in spite of spreading over a wide space. It can be realized by a single photodetector. In addition, the intensity of characteristic luminescence generated in cells that have undergone processing such as marking is low, and the intensity of light obtained by condensing them is still low, so it is detected and the result is output The photodetector (the photodetector set 98 or a part thereof) is preferably a photomultiplier tube. As is known in this technical field, a photomultiplier tube is a tube that cascade-amplifies an electron current by a multi-stage high voltage applied to a cathode group to actually increase a signal gain. In order to further improve the signal-to-noise ratio, the optical path in the signal detector 90 may be blocked from the outside (for example, housed in a container) to substantially reduce noise due to leaked light. The detection result by the signal detector 90 is processed by the transmission-destination electronic control unit 80 and appropriately displayed on the display 100.

  Those skilled in the art (so-called those skilled in the art) can modify the signal detector 90 to suit a particular imaging environment or application by adding, removing or replacing its components. For example, if the signal-to-noise ratio characteristics to be realized are different from those described above, the photodetector set 98 can also be realized by a photodiode. Similarly, in the above example, one light detector set 98 and one set of focusing optical systems 92 and 96 are used to collect the light emitted from the light emission side opening 52 on the light detector set 98. However, the light detector set 98 may be used in an array to cover the opening surface of the emission side opening 52 in terms of area. In that case, the focusing optical system becomes unnecessary.

  In the configuration described above, the incident side opening 48 is arranged above the sample 12 so as to collect the induced radiation. For example, as shown in FIG. 4, the fiber bundle first end portion 42 ′ is arranged. In addition, the specimen 12 'may be captured from the lower side in the field of view of the entrance side opening 48', that is, the specimen 12 'may be captured through the surface of the slide 16' on the side opposite to the smear 14 'side. Is possible. In this case, since the entrance side opening 48 ′ captures the smear 14 ′ in the visual field (within the range of the collection angle θ) through the slide 16 ′, the slide 16 ′ has a characteristic light emission from the cell (of the broken line 82 ′). Use a material that can transmit the material that is transmitted downward). Further, if a light shielding filter 110 such as a bandpass filter that absorbs the radiation beam 64 such as a laser beam is provided so as to cover the slide 16 ′, particularly the surface under the smear 14 ′, the radiation beam 64 is transmitted below the slide 16 ′. Thus, the phenomenon of being incident on the incident side opening 48 'can be suppressed. In the configuration shown in FIG. 4, a partial cylindrical reflector 112 is also provided as an additional member. The light reflected by the partially cylindrical reflector is generally focused on a certain straight line, and the reflector 112 also has such a focusing line. One of the features of the reflector 112 is that it has a focusing line that substantially coincides with the linear locus 74 ′ formed on the smear 14 ′ by the radiation beam 64. By performing imaging using the partial cylindrical reflector 112 having such a focusing line, some of the characteristic light emission 82 ′ directed upward can be reflected in the direction of the linear locus 74 ′. It is possible to increase the amount of feature light emission and thus improve the signal-to-noise ratio. It will be appreciated that a reflector similar to the partial cylindrical reflector 112 can be used with the configuration shown in FIG.

  In the above-described configuration, it is beneficial to use a plurality of types of probes that emit light at different wavelengths (for example, emit fluorescence) from other probes as probes that are used for cell modification and are directly scanned, such as fluorescent probes. There are many cases. If a plurality of types of probes are used successfully, for example, a plurality of types of characteristics of a cell can be measured simultaneously, and noise and false images can be identified and removed. For efficient excitation, it is desirable to provide a radiation source such as a laser light source for each probe type, and in particular, the wavelength of the radiation beam from the radiation source such as a laser beam corresponding to the radiation beam is absorbed by the probe. The wavelength is adapted to the above. A configuration in which a plurality of types of probes are excited by the same radiation source and the induced radiation from these probes is measured can also be suitably employed.

  The use of a plurality of excitation radiation sources helps to suppress interprobe aggregation artifacts that can occur when cells are labeled with multiple types of labels. For example, suppose here are two types of probes that emit stimulated radiation at substantially different wavelengths: a short-wavelength emitting probe that emits shorter-wavelength stimulated radiation and a long-wavelength radiation probe that emits longer-wavelength stimulated radiation. The wavelength of the stimulated radiation is substantially different means that the process of separating the induced radiation from the cell group in which the radiation from the short wavelength radiation probe and the radiation from the long wavelength radiation probe are mixed in accordance with the probe type is separated. This means that the induced radiation can be implemented by passing it through a conventional radiation filter. When attaching such a label to a cell, a particular problem is that if only one radiation source having a wavelength close to short-wavelength stimulated radiation is used as an excitation radiation source, for example, a laser light source, the wavelength of the radiation source is increased. The stimulated emission by the near short wavelength radiation probe can be efficiently caused, but the induced radiation by the long wavelength radiation probe cannot be efficiently caused. If a radiation source with a wavelength closer to that of the stimulated radiation is used, such a stimulated radiation can be caused more efficiently. Of course, it is possible to induce stimulated emission at a suitable excitation-to-radiation ratio in a configuration in which multiple types of probes are excited by a single radiation source. Will also need to be mixed in at a high concentration. When the concentration of the probe is increased, the excitation-to-radiation ratio varies and further aggregation can occur. Since it is sufficient to increase the excitation efficiency even at a low concentration, it would be meaningful to use a separate radiation source such as a laser light source for each probe.

  FIG. 5 shows an example in which a specimen including two types of probes is examined using two laser light sources in connection with the above problem. This figure shows the relationship of the transfer function value (%) 130 with respect to the wavelength 132 in the wavelength range of interest. In the figure, the wavelength represented by the vertical straight line 134 is the wavelength of the first laser light source of the two laser light sources used, and is 488 nm in this example. In this example, a FITC (Fluoresceinisothiocyanate isomer-I) probe having a radiation intensity peak wavelength of about 520 nm as shown in the drawing is used as the first probe. The difference between the first laser wavelength 134 and the intensity peak wavelength on the first probe radiation characteristic 136 matches or matches a deviation known in the art as a Stokes shift. Therefore, this FITC probe can be suitably excited by this laser beam having a wavelength = 488 nm. The Stokes shift is the difference between the absorbed quantum wavelength and the emitted quantum wavelength. Since there is an energy conservation law, the emission wavelength ≧ incident wavelength as shown in this figure, and the difference between the incident wavelength and the emission wavelength is absorbed as thermal energy in the atomic lattice of the substance. A curve 138 in the figure is a preferred example of the characteristics of the first radiation filter provided corresponding to the first laser light source. This first radiation filter characteristic 138 can remove or suppress unnecessary light and the like and various unnecessary frequency components that are reflected by the sample and arrive at the signal detector from the light emitted from the first laser light source. The first probe is designed to substantially transmit light emitted from the first probe, for example, fluorescence. In the illustrated example, the pass band is in the range of about 505 to 545 nm.

  At this time, if an R-PE (R-Phycoerythrin) probe whose intensity peak wavelength in the probe radiation characteristic 140 is 576 nm is used as the second probe used in combination with the first probe which is the FITC probe, there are several problems. Will occur. First, the first probe radiation characteristic 136 and the second probe radiation characteristic 140 overlap to a considerable extent in the wavelength region of about 550 to 600 nm. Therefore, since signal crosstalk occurs, it is difficult to distinguish radiation from the first probe from radiation from the second probe. In order to alleviate this problem, a second radiation filter having a characteristic 142 as shown in this figure, that is, a characteristic having a pass band over a wavelength range of about 575 to 640 nm may be used. This partially alleviates the problem of signal crosstalk because most of the radiation from the first probe is blocked before the signal detector. However, even with such a radiation filter, a similar amount of crosstalk remains and the same problem remains. That is, since the base of the first probe radiation characteristic 136 is considerably in the pass band of the second radiation filter characteristic 142, the sensitivity and the signal-to-noise ratio of the system are lowered accordingly.

  Here, one of the causes of the remarkable signal loss described above is that only one radiation source such as a laser light source is provided in the system. That is, since the wavelength of the first laser light source is significantly different from the wavelength suitable for causing the stimulated emission by the second probe, the stimulated emission by the second probe is less efficient than the stimulated emission by the first probe. Only occurs in In order to cause the stimulated emission by the second probe more efficiently, for example, in the example of FIG. 5, a second laser light source having a radiation wavelength 144 of about 532 nm is used simultaneously with the first laser light source according to the wavelength 134. Good. However, in such cases, new problems that cannot be overcome arise. That is, since the wavelength 144 of about 532 nm of the second laser light source is in the pass band on the first radiation filter characteristic 138, when the laser light from the second laser light source is reflected by the sample or the like, the reflected light is reflected to the first probe. May be misdetected as if it were stimulated radiation from.

FIG. 6 shows a configuration of an apparatus according to the first reference example of the present invention. The apparatus shown in this figure is provided with a plurality of signal detectors and laser light sources, and the time-multiplexed operation of the laser light sources causes the above-mentioned problem, that is, when a plurality of probes are used. This device eliminates the problems that occur. In this reference example , stimulated emission from the specimen is caused by a plurality of laser wavelengths. However, in this embodiment, stimulated emission is caused at the same time for the plurality of laser wavelengths as in the example described above. Instead, it is caused to occur in turn or alternately. Therefore, there is no problem due to the overlap between the radiation spectrum range of one probe and the wavelength of the excitation radiation source related to another probe. In addition, for example, laser light that causes stimulated emission in the first probe hardly causes stimulated emission by the second probe, so that light that is induced and emitted from the first probe cannot be distinguished from light that is induced and emitted from the second probe. The phenomenon that light reception is detected and processed, that is, signal crosstalk is suppressed. In contrast, in the previous example in which the operation of the laser light source is not time-division multiplexed, emission from the first probe by induction (characteristic 136 in FIG. 5) and emission from the second probe by induction (140). In general, a dichroic mirror having a transfer characteristic as indicated by a curve 146 in FIG. As can be seen from FIG. 5, a considerable portion of the first probe radiation characteristic 136 is unnecessarily intruded into the passband of the transmission characteristic 146 of this dichroic mirror, for example, at a wavelength longer than about 520 nm.

In the present reference example shown in FIG. 6 for simplification of understanding, for example , a first laser light source 62 ′ that emits a 488 nm laser beam 64 ′ and a 532 nm laser beam 64 ″ are emitted as radiation sources. A second laser light source 62 '' is provided. The first dichroic mirror 150 receives these laser beams 64 ′ and 64 ″ and emits them as a radiation beam 64 for excitation in a wavelength selective manner. Between the laser light sources 62 ′ and 62 ″ and the first dichroic mirror 150, shutters 148 ′ and 148 ″ are provided corresponding to the laser light sources, respectively. The signal is incident on the first dichroic mirror 150 through the corresponding one of the shutters 148 ′ and 148 ″. In the shutters 148 ′ and 148 ″, the radiation beam 64 emitted from the first dichroic mirror 150 always includes only the laser light 64 ′ or 64 ″ from any laser light source 62 ′ or 62 ″. So that they work together. The radiation beam 64 is reflected by the reflector 152 toward the galvanometer 66. The galvanometer 66 scans the specimen 12 with the reflected radiation beam 64. A focusing lens set 70 may be provided between the galvanometer 66 and the specimen 12. The galvanometer 66 and the focusing lens set 70 operate in substantially the same manner as described above with reference to FIG.

  Fluorescence stimulated and emitted from the first and second probes in the specimen 12 is received by the optical path 154 and transmitted to the first lens 92 via the optical path 154. Since the optical path 154 is shaped, the light is converged and condensed into a beam shape by this transmission. The convergent light collimated by the first lens 92 is appropriately wavelength-selectively split by the second dichroic mirror 156, and the split light is obtained by the corresponding one of the photodetector sets 98 ′ and 98 ″. Received light. In the example of this figure, a light shielding filter 94 ′ and a second lens set 96 ′ are provided in front of the light detector set 98 ′, and a light shielding filter 94 ″ and a second lens are provided in front of the light detector set 98 ″. Each set 96 '' is arranged. The functions of the light shielding filter and the second lens set are substantially the same as those described above. The fluorescence coming from each probe via these is detected by the corresponding one of the photodetector sets 98 ′ and 98 ″, which detects the corresponding radiation beam 64 ′ or 98 ′ or 98 ″. The control unit 80 informs the control unit 80 of the intensity level detected by receiving and detecting the optical signal 64 ″, and the control unit 80 scans by a radiation source and a scanner, a motor so as to obtain a pixel array representing at least a part of the sample 12. The detection result is processed in association with, for example, a radiation beam while coordinating the specimen movement and detection by the photodetector set. The specimen movement may be a linear movement (see FIG. 1) in a direction perpendicular to the beam trajectory, or may be a movement accompanied by rotation around an axis perpendicular to the surface of the specimen 12. In the former case, the incident side opening is arranged substantially parallel to the beam locus, and in the latter case, the incident side opening is arranged substantially parallel to the radial direction. Furthermore, a single photodetector set may be provided in place of the photodetector sets 98 'and 98 ". In that case, for example, in place of the light shielding filters 94 ′ and 94 ″ provided for each photodetector set, the light shielding filters 94 ′ and 94 ″ are incorporated in a single rotating disk, and the photodetector set. The disk may be rotated in synchronism with the operation of the laser beam shutters 148 'and 148' '. That is, when the fluorescence related to the first laser light source 62 ′ arrives, the fluorescence passes through the light shielding filter 94 ′, and when the fluorescence related to the second laser light source 62 ″ arrives, the fluorescence passes through the light shielding filter 94 ″. Thus, the disk may be operated in synchronization with the shutter.

FIG. 7 shows the configuration of an apparatus according to the second reference example of the present invention. The apparatus shown in this figure is also an apparatus that has solved the above-mentioned problems as in the first reference example . In this apparatus, the total scanning time can be further shortened. That is, also in this reference example , since the stimulated emission in the specimen 12 is caused to occur in order or alternately for each radiation beam, not for all radiation beams simultaneously, the above-described problems can be solved. In this reference example , since this is realized using the rotating polygon scanner set 160, high-speed scanning is possible. The rotating polygon scanner set 160 includes a polyhedral mirror 160a, a motor 160b for rotating the polyhedral mirror 160a, and a flywheel 160c, as shown in FIG. The motor 160b and the flywheel 160c are selected and arranged so that the polyhedral mirror 160a can be smoothly rotated, that is, optical signal jitter due to reverse fine movement of the reflecting surface does not occur. Unlike a galvanometer, reverse fine movement hardly occurs even when it is rotated at a high speed, and therefore jitter hardly occurs. As shown in the figure, the rotating polygon scanner set 160 scans the surface of the specimen 12 with a radiation beam 64 such as a laser. That is, when the laser beam 64 ′ from the first laser light source 62 ′ arrives at the rotating polygon scanner set 160 as a radiation beam 64, the radiation beam 64 is angularly scanned by a certain surface of the polyhedral mirror 160 a and applied to the surface of the specimen 12. Incident. When the laser beam 64 ″ from the second laser light source 62 ″ arrives at the rotating polygon scanner set 160 as a radiation beam 64, the radiation beam 64 is angularly scanned by another surface of the polyhedral mirror 160a, and the specimen 12 is scanned. Incident on the surface. Therefore, according to this reference example , it is possible to implement a detection method in which stimulated emission is caused in all the probes by laser light from a certain laser light source and the induced emission is detected without laser interference. For example, when two types of probes are used, when the laser beam 64 ′ from the first laser light source 62 ′ is irradiated as the radiation beam 64, the radiation beam 64 causes both the first and second probes to emit stimulated emission and detect them. On the other hand, when the laser beam 64 ″ from the second laser light source 62 ″ is irradiated as the radiation beam 64, the radiation beam 64 causes stimulated emission in only one of the first and second probes. For example, it is detected. In this reference example , only one photodetector (92, 94, 96 and 98) is provided one by one as shown in the figure. In order to perform time division multiplex detection under this configuration, for example, a time division multiplex operation filter 162 that is inserted in synchronization with the shutters 148 ′ and 148 ″ or whose characteristics are switched (for example, filter elements are switched) is provided. Good. However, the configuration using the time division multiplex filter 162 is only an example configuration, and thus a configuration in which a plurality of photodetectors are included in the configuration shown in FIG. 7 is also included in the technical scope of the present invention.

Figure 8 shows the structure of a device according to the implementation embodiments of the present invention. Similarly to the first and second reference examples, the apparatus shown in this figure is an apparatus that has solved the above-mentioned problems. Also in this embodiment, since the stimulated emission in the specimen 12 is caused to occur sequentially or alternately for each radiation beam, not simultaneously with all radiation beams, the above-described problems can be solved. Also in this embodiment, the rotating polygon scanner set 160 is used as means for scanning the surface of the specimen 12 with a radiation beam such as a laser beam, and the principle of scanning of the radiation beam by the rotation of the rotating polygon scanner set 160 is as follows. This is the same as the reference example shown in FIG. However, in this embodiment, it is formed by a radiation beam from the other radiation source (for example, the laser light source 62 ″) between the scanning lines formed by the radiation beam from one radiation source (for example, the laser light source 62 ′). Since the scanning line to be inserted is automatically inserted, the scanning speed can be doubled as compared with the reference example shown in FIG. 7 without increasing the rotational speed of the rotating polygon scanner set 160. That is, in the present embodiment, by appropriately setting the incident angle from the laser light sources 62 ′ and 62 ″ to the rotating polygon scanner set 160, a scanning line is inserted into the rotating polygon scanner set 160 in addition to sequential or alternate scanning. Is done automatically. Moreover, it is not necessary to use a shutter in either of the laser light sources 62 ′ and 62 ″. More specifically, by appropriately arranging the laser light sources 62 ′ and 62 ″ in a predetermined positional relationship with respect to the incident side opening, the rotation of the rotating polygon scanner set 160 in the direction of the arrow in the figure is changed. The radiation beam from one laser light source 62 ′ is angularly scanned by reflection on each surface of the rotating polygon scanner set 160, and the radiation beam from the second laser light source 62 ″ is reflected on each surface of the rotating polygon scanner set 160 so as to follow this. The angle scan is performed by. Since the scanning lines are inserted by scanning each surface of the rotating polygon scanner set 160, the number of scanning lines is effectively doubled. It should be noted that this idea of appropriately arranging the laser light sources so that the scanning lines are inserted can be adopted even in a configuration in which the number of laser light sources is increased. Also, the number of surfaces of the rotating polygon scanner set 160 can be determined as desired.

The embodiment shown in FIG. 8 is particularly useful in the case where the scanning efficiency is less than 50%, that is, there is room for doubling the efficiency by scanning line automatic insertion. One advantage of this system or approach is that additional scan lines can be inserted without increasing the rotational speed of the rotating polygon scanner set 160 or reducing the overall processing speed. In addition to this, the galvanometer 66 used in the above-mentioned reference example generates jitter due to the vibration, while the rotating polygon scanner set 160 hardly generates such jitter. There are also advantages. Furthermore, since the embodiment shown in FIG. 8 has the ability to detect twice the amount of information within the time spent for one scan in another reference example , the total time required for the scan Is significantly shortened. It should be appreciated that the arrangement and order of components in FIG. 8 are merely examples, and therefore reference examples with modifications thereof are also included in the technical scope of the present invention.

In any of the reference examples and embodiments shown in FIGS. 6 to 8, the problem of the deterioration of the fluorescence signal due to the overlap with the laser frequency is basically solved. In addition, signal crosstalk is caused by stimulated emission from each probe at a laser frequency at which the radiation efficiency of the target probe can be maximized and the radiation efficiency of other probes causing crosstalk is reduced. Can be reduced. Moreover, in any reference example and embodiment, not only can the cost of the filtering system be remarkably suppressed, but also the filtering capability in the multi-probe multi-laser fluorescence detection system can be significantly improved. According to the embodiment shown in FIG. 8, the number of scanning lines can be doubled as compared with the reference example shown in FIG. 7, and the rotational speed or scanning speed of the rotating polygon scanner set 160 is increased. Can be achieved without any trouble.

In addition, as an effect of the time division multiplex scanning in each reference example and embodiment, there is an effect due to the relationship with the autofluorescence frequency. Noise due to autofluorescence is at a high level in the vicinity of the excitation light wavelength, but is at a lower level at a wavelength far from the excitation light wavelength, such as a stimulated emission wavelength. In the configuration in which the first probe, for example, the FITC probe and the second probe, for example, the R-PE probe, are simultaneously excited and fluorescent, the crosstalk due to the fluorescence from the second probe is removed by filtering as shown in FIG. Therefore, it was necessary to design the first radiation filter characteristic 138. The self generated in the vicinity of the peak of the stimulated emission from the first probe by the radiation beam from the second radiation source even though a considerable component on the long wavelength side of the stimulated radiation 136 related to the first probe is thereby blocked. Noise due to fluorescence could not be suppressed. On the other hand, in the configuration in which time division multiplex scanning is performed, basically no crosstalk occurs, so that the signal-to-noise ratio can be improved without blocking the long wavelength component of the induced radiation from a certain probe. This is useful in connection with image filtering processing.

As mentioned above, although the reference example and embodiment of this invention were demonstrated by the application example especially to cell identification, the thought which concerns on this invention is applicable also to uses other than such a use. For example, the imager or the imaging apparatus having the configuration shown in FIGS. 6 to 8 can be suitably used in various imaging fields where an object to be identified or determined is present. One example of a field that can be used is the biomedical field. In particular, it can be suitably used for an array in which 10 to 10000 DNA molecules are arranged, that is, an array known as a DNA chip in this technical field. When the present invention is implemented for a DNA chip, for example, the tag is removed from the inside of a DNA chip that contains a large number of DNA molecules treated so that a fluorescent tag is selectively incorporated into a group of molecules. In identifying the inserted DNA molecule, the apparatus according to embodiments of the present invention can be used publicly. If the present invention is implemented in such a field, an imaging apparatus capable of accessing a sample at a speed several times faster than that which can be realized by conventional techniques can be obtained.

1 is a perspective view illustrating an imaging apparatus suitable for incorporating an apparatus according to an embodiment of the present invention. It is an expansion perspective view which shows the relationship between the shaping optical fiber bundle which comprises the imaging apparatus shown in FIG. 1, and a sample. FIG. 2 is an enlarged end view showing one form of a first end portion forming a shaping optical fiber bundle constituting the imaging apparatus shown in FIG. FIG. 7 is an enlarged end view showing another form of the first end portion forming the shaping optical fiber bundle constituting the imaging apparatus shown in FIG. 1, in particular, the incident side opening. It is the figure which plotted excitation light and the induced radiation produced by it. It is a schematic diagram which shows the apparatus structure and arrangement | positioning in the 1st reference example of this invention. It is a schematic diagram which shows the apparatus structure and arrangement | positioning in the 2nd reference example of this invention. Is a schematic view showing an apparatus constructed and arranged in the implementation of the invention.

Explanation of symbols

12, 12 ′ specimen, 14, 14 ′ biological smear, 20 imager stage, 62 ′, 62 ″ radiation source (laser light source), 64, 64 ′, 64 ″ radiation beam (laser light / excitation light / excitation field) , 80 processor (electronic control unit), 98, 98 ′, 98 ″ signal detector light detector, 154 light path.

Claims (4)

An imager stage that supports the specimen,
An optical path having an entrance aperture positioned proximate to the sample to capture the sample on the imager stage within its field of view and an exit aperture away from it;
A plurality of radiation sources that emit placed by and respectively the radiation beam is divided into a plurality based on the input elevation side opening,
Has two or more reflective surfaces, each reflective surface is rotating polygon scanner査run on selected areas of each radiating specimen reflects radiation beams incident from the radiation source corresponding to the respective reflecting surfaces When,
Light detection that occurs due to the action of the radiation beam on the specimen, is received at the entrance-side aperture, transmitted to the exit-side aperture via the optical path, and received and detected by associating the light signal emitted from the exit-side aperture with each radiation source Instrument system,
A processor for processing the optical signal detected by the photodetector system in association with each radiation source;
A scanning imager for specimen imaging comprising :
Each radiation source emits a radiation beam reflected by each reflecting surface of the rotary polygon scanner corresponding to each radiation source, and the position of the specimen in which each reflected radiation beam is shifted in a direction perpendicular to the scanning direction of each radiation beam. Can be scanned in order by shifting the action time of each radiation beam on the sample, so that the angle of incidence on the rotary polygon mirror increases the number of sample scans per rotation of the rotary polygon scanner. It is arranged in a predetermined positional relationship with the rotary polygon mirror,
A scanning imager for specimen imaging characterized by the above .   The scanning imager for specimen imaging according to claim 1, wherein the light signal is generated from a plurality of types of fluorescent probes that emit fluorescence at different wavelengths.   3. The specimen imaging scanning imager according to claim 2, wherein a plurality of optical signals generated by the fluorescent probe are received and detected by the photodetector system as non-coherent optical signals that do not interfere with each other. Scanning imager for imaging. An imager stage that supports the sample, an optical path having an incident-side aperture positioned close to the sample and an exit-side aperture away from the sample so as to capture the sample on the imager stage in its field of view, and the incident-side aperture as a reference And a plurality of radiation sources each of which emits a radiation beam and two or more reflection surfaces, and each reflection surface reflects a radiation beam incident from each radiation source corresponding to each reflection surface. A rotating polygon scanner that scans each radiation over a selected area of the specimen and the radiation beam acting on the specimen, and is received by the incident side aperture and transmitted to the exit side aperture via the optical path. An optical detector system that detects and detects the light signal emitted from the side opening in association with each radiation source, and associates the optical signal detected by the optical detector system with each radiation source A scanning imager for specimen imaging comprising a processor for processing, wherein each radiation source is reflected by each reflecting surface of a rotary polygon scanner corresponding to each radiation source, and each radiation beam is scanned by each radiation beam. Rotating type that can scan the position of the sample shifted in the direction perpendicular to the direction in order by shifting the working time of each radiation beam on the sample and increasing the number of sample scans per rotation of the rotating polygon scanner Preparing a scanning imager for sample imaging arranged in a predetermined positional relationship with a rotary polygon mirror so as to have an incident angle on the polygon mirror;
Reflecting radiation from a plurality of radiation beams on each reflecting surface of the rotary polygon scanner corresponding to each radiation source, and increasing the number of scans per rotation of the rotary polygon scanner;
On specimen along a linear path, shifting the action time for specimens of each radiation beam a plurality of radiation beams reflected respectively having different radiation wavelengths position of the sample shifted in the scanning direction perpendicular to the direction of the radiation beam Step to sequentially sweep,
Collecting light generated by the action of each radiation beam on the specimen;
Transmitting the collected light to a predetermined emission location by an optical path;
Detecting the collected light in association with the radiation beam at the exit location;
A step of performing a sweep of the sample in a raster manner in order with a plurality of radiation beams by moving the sample in a direction substantially orthogonal to the linear sweep path by the radiation beam while coordinating with the sweep by the radiation beam,
Generating a pixel array representing at least a part of the sample in association with the radiation beam by coordinating the sweep operation, the movement operation, and the detection operation;
An imaging method comprising: